PROJECT SUMMARY With the success of checkpoint blockade immunotherapy, it is well accepted that the immune system shapes patient outcomes in cancer. Targeting either cytotoxic T-lymphocyte associated protein 4 (CTLA-4) or programmed cell death protein 1 (PD1) demonstrates that reinvigoration of cytotoxic T cells is essential for a clinical response. Despite the success of immuno-oncology strategies, a sizeable proportion of patients fail to respond to treatment, thus highlighting the importance of novel combinatorial strategies. Regulatory T cells (Tregs), marked by the master transcription factor forkhead box P3 (FOXP3), are potent suppressors of inflammatory processes. Though essential for maintenance of peripheral immune homeostasis and prevention of autoimmunity, it is well established that the suppressive function of Tregs is a major barrier to anti-tumor immunity, contributing to disease progression and failure of current immunotherapeutic agents. Several lines of evidence suggest that targeting Neuropilin-1 (NRP1, CD304), a transmembrane coreceptor for semaphorin-4a among other ligands, may decrease Treg inhibitory function in malignancy. In fact, deletion of the Nrp1 gene in murine Tregs diminishes Treg suppressive function in the tumor without autoimmune consequences. Intratumoral NRP1-deficient Treg dysfunction was potentiated by their adoption of a pro-inflammatory phenotype, including marked production of potent proinflammatory cytokine Interferon-gamma (IFN?). The function of NRP1 on human Tregs has never before been studied in the context of cancer. Therefore, it remains to be determined (1) what signals drive NRP1 expression and surface trafficking in cancer patients and (2) what is the functional consequence of NRP1 expression for human Tregs. Furthermore, the direct impact of proinflammatory mediators on Tregs in cancer patients has not been fully characterized. We hypothesize that activated Tregs integrate signals from both the periphery and the tumor microenvironment (TME), thus upregulating NRP1 in response to a proinflammatory cytokine milieu and increasing surface expression following contact-dependent activation cues. It remains to be determined if NRP1 is required for optimal Treg function in cancer patients. We are testing whether pharmacologic blockade of NRP1 ligation restricts human NRP1+ Treg suppressive advantage. We are also investigating if lack of NRP1 expression on intratumoral Tregs will determine the success of the inflammatory response to anti-PD1 immunotherapy in head and neck squamous cell carcinoma. This proposal will identify drivers of NRP1 expression, detail the importance of NRP1 expression on tumor associated Tregs, and evaluate whether high NRP1 expression is associated with immunotherapy failure. If successful, these studies may provide the preclinical rationale for exploring the combination of NRP1 inhibitors with current immunotherapy agents.